The liver is the only internal organ capable of natural regeneration of large portion's of lost tissue. As little as one third of a liver can regenerate itself in vivo (Michalopoulos, 2010, Am J Pathol, 176:2-13). However, liver cells placed in culture rapidly lose their in vivo phenotypic characteristics and functional abilities. This situation has limited the ability to study regenerative properties and basic functions of liver cells in vitro. As shown previously, it is difficult to maintain liver-specific function of primary hepatocytes in culture for more than one week without an adequate supportive microenvironment including substrates, e.g. extracellular matrix (ECM) coatings or feeder layers (Dunn et al., 1989, FASEB J, 3:174-7). The lack of a suitable culture platform has restricted drug discovery, toxicology, cancer, and tissue regeneration studies for liver cells. Despite decades of research, current culture products are not suitable for the expansion and maintenance of the highly specialized functions of hepatocytes. While some generic products and systems are available, they do not meet the specific culture requirements of primary liver cells. Thus, there is a need for cell culture systems that mimic the in vivo characteristics of hepatocytes.
Elements that influence human hepatocyte cultures include cell-ECM interactions, soluble growth factors and cytokines, physical factors (e.g. stress and strain) (Sadoshima et al., 1997, Annu Rev Physiol, 59:551-71; Syedain et al., 2008, Proc Natl Acad Sci USA, 105:6537-42), and cell-cell communications (Funderburgh et al., 2008, Mol Vis, 14:308-17). Importantly, cell-ECM interactions play a fundamental role in hepatocyte growth (Apte et al., 2009, Hepatology, 50:844-51; Hammond et al., 2011, J Hepatol, 54:279-87), liver organ development (Hanley et al., 2008, J Biol Chem, 283:14063-71; Semler et al., 2006, Adv Biochem Eng Biotechnol, 102:1-46), tissue regeneration (Apte et al., 2009, Hepatology, 50:844-51; Jun et al., 2010; Aging, 2:627-31; Tai et al., 2010, Biomaterials, 31:48-57), wound healing (Jun et al., 2010; Aging, 2:627-31; Gilbert et al., 2009, Laryngoscope, 119:1856-63; Povero et al., 2010, Histol Histopathol, 25:1075-91) and malignancy (Hanley et al., 2008, J Biol Chem, 283:14063-71; Mon et al., 2009, Methods Mol Biol, 512:279-93). The liver ECM contains proteins and carbohydrates that provide support and anchorage for cells, segregate tissues, and regulate intercellular communication. Commercially available tissue extracts enriched in matrix (e.g. Matrigel, collagen-I, extracts from amnions) have been used successfully as culture substrata for many years (Yamasaki et al., 2006, J Hepatol, 44:749-57). However, they are not tissue-specific (Everitt et al., 1996, J Leukoc Biol, 60:199-206; Shirahashi et al., 2004, Cell Transplant, 13:197-211). It was reported that tissue-derived ECM could be used successfully as a 2D substrate for the cell type that originated from that tissue (Zhang et al., 2009, Biomaterials, 30:4021-8). Additionally, a 3D co-culture system using a porous ECM scaffold combined with dynamic culture conditions promoted the formation of a multilayered urothelium and infiltration of smooth muscle cells into the matrix. This construct could be used to engineer urological tissue for bladder or urethral tissue reconstruction (Liu et al., 2009, Biomaterials, 30:3865-73).
Conventional tissue culture techniques relate to growing cells in vitro. Usually, the cells are cultured on a coated surface having a negative charge to enhance the attachment and sometimes proliferation of mammalian cells in culture. However, traditionally it has been most difficult to achieve a satisfactory attachment, maintenance, and propagation of mammalian cells using conventional tissue culture surfaces. Improvements have been made by adding layers of collagen gel or depositing an extracellular matrix onto the tissue culture plates and dishes to facilitate cellular attachment and proliferation. These techniques, however, are hindered by the shortcoming that the cultured cells often lose their function and viability.
Tissue engineered products hold immense potential for treating and curing disease. However, due to the complex nature of constructs comprised of living cells, biomaterials, and soluble factors, current regulatory practices and the need for long validation studies impede the clinical and commercial success of such products. One solution is the application of tissue-engineered products in commercial in vitro settings that both yield financial gain for industry while providing platforms for research, testing, and validation. The use of human cells for efficacy and toxicology screening of potential pharmaceutical agents is a current practice, but more complex human tissue constructs might yield better results that are more clinically relevant. Engineered human tissues have the potential to screen drug candidates quicker and more inexpensively in comparison to animal studies, while returning desirable results that are more relevant to humans. Incorporation of such practices in the early phases of drug development may successfully bridge the gap between laboratory research and clinical application of tissue-engineered products, both scientifically and financially (Greenhough, et al., 2010, Toxicology 278(3): 250-255). To that end, there is a need for a new biomaterial system for preparing and maintaining human hepatocyte tissue constructs with potential to be used as a drug and toxicology screening tool.
Despite many years of tissue culture research, no techniques are currently available to expand and maintain the function of highly specified cells. While generic two-dimensional (2-D) and three-dimensional (3-D) coatings exist, these products are not specific to cells. The present invention fills this gap in the art.